Diagnostic performance and comparison of ultrasensitive and conventional rapid diagnostic test, thick blood smear and quantitative PCR for detection of low-density Plasmodium falciparum infections during a controlled human malaria infection study in Equatorial Guinea.

BACKGROUND: Progress towards malaria elimination has stagnated, partly because infections persisting at low parasite densities comprise a large reservoir contributing to ongoing malaria transmission and are difficult to detect. This study compared the performance of an ultrasensitive rapid diagnostic test (uRDT) designed to detect low density infections to a conventional RDT (cRDT), expert microscopy using Giemsa-stained thick blood smears (TBS), and quantitative polymerase chain reaction (qPCR) during a controlled human malaria infection (CHMI) study conducted in malaria exposed adults (NCT03590340). METHODS: Blood samples were collected from healthy Equatoguineans aged 18-35 years beginning on day 8 after CHMI with 3.2 × 103 cryopreserved, infectious Plasmodium falciparum sporozoites (PfSPZ Challenge, strain NF54) administered by direct venous inoculation. qPCR (18s ribosomal DNA), uRDT (Alere™ Malaria Ag P.f.), cRDT [Carestart Malaria Pf/PAN (PfHRP2/pLDH)], and TBS were performed daily until the volunteer became TBS positive and treatment was administered. qPCR was the reference for the presence of Plasmodium falciparum parasites. RESULTS: 279 samples were collected from 24 participants; 123 were positive by qPCR. TBS detected 24/123 (19.5% sensitivity [95% CI 13.1-27.8%]), uRDT 21/123 (17.1% sensitivity [95% CI 11.1-25.1%]), cRDT 10/123 (8.1% sensitivity [95% CI 4.2-14.8%]); all were 100% specific and did not detect any positive samples not detected by qPCR. TBS and uRDT were more sensitive than cRDT (TBS vs. cRDT p = 0.015; uRDT vs. cRDT p = 0.053), detecting parasitaemias as low as 3.7 parasites/µL (p/µL) (TBS and uRDT) compared to 5.6 p/µL (cRDT) based on TBS density measurements. TBS, uRDT and cRDT did not detect any of the 70/123 samples positive by qPCR below 5.86 p/µL, the qPCR density corresponding to 3.7 p/µL by TBS. The median prepatent periods in days (ranges) were 14.5 (10-20), 18.0 (15-28), 18.0 (15-20) and 18.0 (16-24) for qPCR, TBS, uRDT and cRDT, respectively; qPCR detected parasitaemia significantly earlier (3.5 days) than the other tests. CONCLUSIONS: TBS and uRDT had similar sensitivities, both were more sensitive than cRDT, and neither matched qPCR for detecting low density parasitaemia. uRDT could be considered an alternative to TBS in selected applications, such as CHMI or field diagnosis, where qualitative, dichotomous results for malaria infection might be sufficient.

Quantification of Plasmodium knowlesi versus Plasmodium falciparum in the rhesus liver: implications for malaria vaccine studies in rhesus models.

BACKGROUND: Rhesus macaques are valuable pre-clinical models for malaria vaccine development. The Plasmodium knowlesi/rhesus and Plasmodium falciparum/rhesus models are two established platforms for malaria vaccine testing, and both have previously been used to assess live-attenuated sporozoite vaccines. However, there is evidence that the susceptibility of the rhesus liver to P. knowlesi versus P. falciparum sporozoites likely differs, potentially complicating comparisons between these two platforms. METHODS: To quantify the differing susceptibility of rhesus to P. knowlesi and P. falciparum sporozoites, animals were infected by direct venous inoculation of purified, cryopreserved wild-type P. knowlesi sporozoites (PkSPZ) or P. falciparum sporozoites (PfSPZ). The entire liver was collected 5 days post-infection, and parasite burden in each liver lobe was quantified using an ultrasensitive Plasmodium 18S rRNA RT-PCR biomarker assay. The potential of using 18S rRNA copy number in the rhesus liver to directly measure the efficacy of vaccines targeting P. falciparum sporozoites and liver stages was also theoretically evaluated. RESULTS: Infection of rhesus with a high dose of PkSPZ led to consistently high burden liver stage infections (range 9.5-10.1 log10 copies 18S rRNA/g of liver), with similar amounts of parasite 18S rRNA detected in every liver lobe. Inoculation of rhesus with high doses of PfSPZ led to more variable, lower liver burdens (range 4.9-6.6 log10 copies 18S rRNA/g of liver in infected lobes), with parasite 18S rRNA below the limit of detection in some liver lobes. The low signal and heterogeneity of liver burden in the PfSPZ-infected animals indicates that even this extremely sensitive molecular assay cannot be used to assess reliably vaccine efficacy in the P. falciparum/rhesus platform. CONCLUSIONS: Detection of 18S rRNA in the liver following high dose intravenous PfSPZ confirmed that rhesus are modestly susceptible to wild-type P. falciparum sporozoites. However, comparison of 18S rRNA RT-PCR biomarker signal indicates that the P. falciparum liver burden was 3-5 logs lower than in PkSPZ-infected animals. Quantification of this difference in liver stage burden will help guide and interpret data from pre-clinical studies of live-attenuated sporozoite vaccines in rhesus models.

Safety, immunogenicity and efficacy of PfSPZ Vaccine against malaria in infants in western Kenya: a double-blind, randomized, placebo-controlled phase 2 trial.

The radiation-attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine provides protection against P. falciparum infection in malaria-naïve adults. Preclinical studies show that T cell-mediated immunity is required for protection and is readily induced in humans after vaccination. However, previous malaria exposure can limit immune responses and vaccine efficacy (VE) in adults. We hypothesized that infants with less previous exposure to malaria would have improved immunity and protection. We conducted a multi-arm, randomized, double-blind, placebo-controlled trial in 336 infants aged 5-12 months to determine the safety, tolerability, immunogenicity and efficacy of the PfSPZ Vaccine in infants in a high-transmission malaria setting in western Kenya ( NCT02687373 ). Groups of 84 infants each received 4.5 × 105, 9.0 × 105 or 1.8 × 106 PfSPZ Vaccine or saline three times at 8-week intervals. The vaccine was well tolerated; 52 (20.6%) children in the vaccine groups and 20 (23.8%) in the placebo group experienced related solicited adverse events (AEs) within 28 d postvaccination and most were mild. There was 1 grade 3-related solicited AE in the vaccine group (0.4%) and 2 in the placebo group (2.4%). Seizures were more common in the highest-dose group (14.3%) compared to 6.0% of controls, with most being attributed to malaria. There was no significant protection against P. falciparum infection in any dose group at 6 months (VE in the 9.0 × 105 dose group = -6.5%, P = 0.598, the primary statistical end point of the study). VE against clinical malaria 3 months after the last dose in the highest-dose group was 45.8% (P = 0.027), an exploratory end point. There was a dose-dependent increase in antibody responses that correlated with VE at 6 months in the lowest- and highest-dose groups. T cell responses were undetectable across all dose groups. Detection of Vδ2+Vγ9+ T cells, which have been correlated with induction of PfSPZ Vaccine T cell immunity and protection in adults, were infrequent. These data suggest that PfSPZ Vaccine-induced T cell immunity is age-dependent and may be influenced by Vδ2+Vγ9+ T cell frequency. Since there was no significant VE at 6 months in these infants, these vaccine regimens will likely not be pursued further in this age group.